Several methods for isolating nucleic acids such as RNA and/or DNA are known in the prior art that are based on different principles. Examples of common nucleic acid isolation methods include but are not limited to extraction, solid-phase extraction, phenol-chloroform extraction, chromatography, precipitation and combinations thereof. Very common are nucleic acid isolation methods which involve the use of chaotropic agents and/or alcohol in order to bind the nucleic acids to a solid phase, e.g. a solid phase comprising or consisting of silica. The isolation of RNA is particularly challenging, because RNAses are omnipresent in rather high amounts and are active over a broad temperature range and usually do not need co-factors for their activity. Therefore, it is a challenge to provide a RNA isolation method which provides the RNA with good yield and quality.
Furthermore, in many fields such as e.g. the diagnostic field it is desirous or even mandatory to isolate the nucleic acids from a large number of samples. For this purpose it is common to use automated processes (wherein e.g. many samples are processed at the same time). To assist the user and to reduce hands-on-time, robotic systems are commonly used that can process a large number of samples in parallel. Usually, the samples are prepared manually for nucleic acid isolation and are then entered into the robotic system. Respective manual preparation steps are e.g. common for stabilised blood samples. Respective manual steps include e.g. the centrifugation of the stabilised sample to generate a nucleic acid containing pellet and the resuspension of the pellet e.g. in a resuspension buffer (thereby reducing the sample volume). The respectively resuspended samples are then ready e.g. for sample digestion and isolation and are placed into the robotic system. Examples of commercially available robotic systems that operate according to this or a similar principle include but are not limited to QIAsymphony (QIAGEN), QIAcube (QIAGEN) and MagnaPure 96 (ROCHE).
Even though these robotic systems provide remarkable advantages when processing a large number of samples, they also have certain limitations. E.g. said robotic systems can usually only process a certain number of samples at one time. Said number is often lower than the number of samples that is manually prepared as one batch for nucleic acid isolation. Thus, not all of the prepared samples can be processed at the same time. This has the effect that the samples prepared for nucleic acid isolation often have different holding times between their preparation for nucleic acid isolation (e.g. the centrifugation and resuspension of the pellet as described above) and the actual nucleic acid isolation. While the first batch of the prepared samples is processed in the robotic system, the other prepared samples are put on hold. It was found that variations in the holding time of the prepared sample can influence the quality of the isolated nucleic acid as well as the nucleic acid yield. During longer holding times, a portion of the nucleic acids can form precipitates which irreversible stick to the container and thus, can not be purified. Furthermore, the integrity of the nucleic acids, in particular of RNA, can be corrupted. Thus, the nucleic acid quantity and/or quality of the second and subsequent batch of the prepared samples that are processed in the robotic systems are often lower. Hence, a longer holding time may reduce the quality and/or the quantity of the isolated nucleic acids. This in particular poses a problem if complex samples are processed, such as e.g. blood or samples derived from blood and/or samples that were stabilised using a specific chemistry. This problem is further aggravated if large sample volumes (e.g. 1.5 ml and more) are processed. This loss in yield and/or integrity can pose problems in particular in sensitive application fields such as e.g. the diagnostic field wherein an uniform nucleic acid isolation with respect to yield and quantity is important and hence, variations due to the used nucleic acid isolation method must be avoided.
Furthermore, methods that use magnetic beads as solid phase for binding and isolating the nucleic acids often show a reduced nucleic acid yield compared to comparable methods that use a nucleic acid binding membrane instead. Therefore, a loss in nucleic acid yield due to the precipitate formation has an even stronger impact on respective methods and system that use magnetic particles as nucleic acid binding solid phase.
Hence, there is a need in the state of the art to provide a nucleic acid isolation protocol which provides comparable high nucleic acid yields and also a high yield of small nucleic acids, even if the holding times vary between samples and also during extended holding times, a high nucleic acid, in particular RNA integrity.
Therefore, it is an object of the present invention to provide an improved method for isolating a nucleic acid, in particular RNA, from a sample, in particular a blood sample. Furthermore, it is the object of the present invention to provide a method that allows the isolation of nucleic acids from a plurality of samples with a comparable quality and/or quantity, even if the holding times between the preparation of the samples for isolation and the actual isolation of the nucleic acids varies.